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The Western diet (WD) and hyperlipidemia are risk factors for vascular disease, dementia, and cognitive impairment. However, the molecular mechanisms are poorly understood. This pilot study investigated the genomic pathways by which the WD and hyperlipidemia regulate gene expression in brain microvessels. Five-week-old C57BL/6J wild type (WT) control and low-density lipoprotein receptor deficient (LDL-R&minus, /&minus, ) male mice were fed the WD for eight weeks. Differential gene expression, gene networks and pathways, transcription factors, and non-protein coding RNAs were evaluated by a genome-wide microarray and bioinformatics analysis of laser-captured hippocampal microvessels. The WD resulted in the differential expression of 1972 genes. Much of the differentially expressed gene (DEG) was attributable to the differential regulation of cell signaling proteins and their transcription factors, approximately 4% was attributable to the differential expression of miRNAs, and 10% was due to other non-protein coding RNAs, primarily long non-coding RNAs (lncRNAs) and small nucleolar RNAs (snoRNAs) not previously described to be modified by the WD. Lipotoxic injury resulted in complex and multilevel molecular regulation of the hippocampal microvasculature involving transcriptional and post-transcriptional regulation and may provide a molecular basis for a better understanding of hyperlipidemia-associated dementia risk.

mRNA 3′ end processing is an essential step in gene expression. It is well established that canonical eukaryotic pre-mRNA 3′ processing is carried out within a macromolecular machinery consisting of dozens of trans-acting proteins. However, it is unknown whether RNAs play any role in this process. Unexpectedly, we found that a subset of small nucleolar RNAs (snoRNAs) are associated with the mammalian mRNA 3′ processing complex. These snoRNAs primarily interact with Fip1, a component of cleavage and polyadenylation specificity factor (CPSF). We have functionally characterized one of these snoRNAs and our results demonstrated that the U/A-rich SNORD50A inhibits mRNA 3′ processing by blocking the Fip1-poly(A) site (PAS) interaction. Consistently, SNORD50A depletion altered the Fip1–RNA interaction landscape and changed the alternative polyadenylation (APA) profiles and/or transcript levels of a subset of genes. Taken together, our data revealed a novel function for snoRNAs and provided the first evidence that non-coding RNAs may play an important role in regulating mRNA 3′ processing.

RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2'-O-methylation (2'-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2'-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2'-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1 inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1 germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1 mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2'-O-Me can be etiological to human disease.

Hyperlipidemia is a risk factor for dementia, and chronic consumption of a Western Diet (WD) is associated with cognitive impairment. However, the molecular mechanisms underlying the development of microvascular disease in the memory centers of the brain are poorly understood. This pilot study investigated the nutrigenomic pathways by which the WD regulates gene expression in hippocampal brain microvessels of female mice. Five-week-old female low-density lipoprotein receptor deficient (LDL-R−/−) and C57BL/6J wild type (WT) mice were fed a chow or WD for 8 weeks. Metabolics for lipids, glucose and insulin were determined. Differential gene expression, gene networks and pathways, transcription factors, and non-protein coding RNAs were evaluated by genome-wide microarray and bioinformatics analysis of laser captured hippocampal microvessels. The WD resulted in differential expression of 2,412 genes. The majority of differential gene expression was attributable to differential regulation of cell signaling proteins and their transcription factors, approximately 7% was attributable to differential expression of miRNAs, and a lesser proportion was due to other non-protein coding RNAs, primarily long non-coding RNAs (lncRNAs) and small nucleolar RNAs (snoRNAs) not previously described to be modified by the WD in females. Our findings revealed that chronic consumption of the WD resulted in integrated multilevel molecular regulation of the hippocampal microvasculature of female mice and may provide one of the mechanisms underlying vascular dementia.

Rhythmic oscillations of physiological processes depend on integrating the circadian clock and diurnal environment. DNA methylation is epigenetically responsive to daily rhythms, as a subset of CpG dinucleotides in brain exhibit diurnal rhythmic methylation. Here, we show a major genetic effect on rhythmic methylation in a mouse Snord116 deletion model of the imprinted disorder Prader–Willi syndrome (PWS). More than 23,000 diurnally rhythmic CpGs are identified in wild-type cortex, with nearly all lost or phase-shifted in PWS. Circadian dysregulation of a second imprinted Snord cluster at the Temple/Kagami-Ogata syndrome locus is observed at the level of methylation, transcription, and chromatin, providing mechanistic evidence of cross-talk. Genes identified by diurnal epigenetic changes in PWS mice overlapped rhythmic and PWS-specific genes in human brain and are enriched for PWS-relevant phenotypes and pathways. These results support the proposed evolutionary relationship between imprinting and sleep, and suggest possible chronotherapy in the treatment of PWS and related disorders., Many genes have oscillating gene expression pattern in circadian centers of the brain. This study shows cortical diurnal DNA methylation oscillation in a mouse model of Prader-Willi syndrome, and describes corresponding changes in gene expression and chromatin compaction.

Functional characterization of the transcriptome requires tools for the systematic investigation of RNA post-transcriptional modifications. 2΄-O-methylation (2΄-OMe) of the ribose moiety is one of the most abundant post-transcriptional modifications of RNA, although its systematic analysis is difficult due to the lack of reliable high-throughput mapping methods. We describe here a novel high-throughput approach, named 2OMe-seq, that enables fast and accurate mapping at single-base resolution, and relative quantitation, of 2΄-OMe modified residues. We compare our method to other state-of-art approaches, and show that it achieves higher sensitivity and specificity. By applying 2OMe-seq to HeLa cells, we show that it is able to recover the majority of the annotated 2΄-OMe sites on ribosomal RNA. By performing knockdown of the Fibrillarin methyltransferase in mouse embryonic stem cells (ESCs) we show the ability of 2OMe-seq to capture 2΄-O-Methylation level variations. Moreover, using 2OMe-seq data we here report the discovery of 12 previously unannotated 2΄-OMe sites across 18S and 28S rRNAs, 11 of which are conserved in both human and mouse cells, and assigned the respective snoRNAs for all sites. Our approach expands the repertoire of methods for transcriptome-wide mapping of RNA post-transcriptional modifications, and promises to provide novel insights into the role of this modification.

Summary To identify endogenous miRNA-target sites, we isolated AGO-bound RNAs from Caenorhabditis elegans by individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP), which fortuitously also produced miRNA-target chimeric reads. Through the analysis of thousands of reproducible chimeras, pairing to the miRNA seed emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3′ sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families. By editing an endogenous target site, we demonstrate that 3′ pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions. Finally, we present a simplified method, chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis revealed that sequences in the 5′ as well as the 3′ regions of a miRNA provide the information necessary for stable and specific miRNA-target interactions in vivo.

Although ribosomes are ubiquitous and essential for life, recent data indicate that monogenic causes of ribosomal dysfunction can confer a remarkable degree of specificity in terms of human disease phenotype. Box C/D small nucleolar RNAs (snoRNAs) are evolutionarily conserved non-protein-coding RNAs involved in ribosome biogenesis. Here we show that biallelic mutations in the gene SNORD118, encoding the box C/D snoRNA U8, cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts (LCC), presenting at any age from early childhood to late adulthood. These mutations affect U8 expression, processing and protein binding and thus implicate U8 as essential in cerebral vascular homeostasis.

Many Plasmodium falciparum proteins do not share homology with, and are generally longer than their respective orthologs. This, to some extent, can be attributed to insertions. Here, we studied a P. falciparum RNA hypermethylase, trimethylguanosine synthase (PfTGS1) that harbors a 76 amino acid insertion in its methyltransferase domain. Bioinformatics analysis revealed that this insertion was present in TGS1 orthologs from other Plasmodium species as well. Interestingly, a classical nuclear localization signal (NLS) was predicted in the insertions of primate parasite TGS1 proteins. To check whether these predicted NLS are functional, we developed an in vivo heterologous system using S. cerevisiae. The predicted NLS when fused to dimeric GFP were able to localize the fusion protein to the nucleus in yeast indicating that it is indeed recognized by the yeast nuclear import machinery. We further showed that the PfTGS1 NLS binds to P. falciparum importin-alpha in vitro, confirming that the NLS is also recognized by the P. falciparum classical nuclear import machinery. Thus, in this study we report a novel function of the insertion in PfTGS1. (C) 2016 Elsevier B.V. All rights reserved.

Fluorosis is caused by excess of fluoride intake over a long period of time. Aberrant change in the Runt-related transcription factor 2 (RUNX2) mediated signaling cascade is one of the decisive steps during the pathogenesis of fluorosis. Up to date, role of fluoride on the epigenetic alterations is not studied. In the present study, global expression profiling of short noncoding RNAs, in particular miRNAs and snoRNAs, was carried out in sodium fluoride (NaF) treated human osteosarcoma (HOS) cells to understand their possible role in the development of fluorosis. qPCR and in silico hybridization revealed that miR-124 and miR-155 can be directly involved in the transcriptional regulation of Runt-related transcription factor 2 (RUNX2) and receptor activator of nuclear factorκ-B ligand (RANKL) genes. Compared to control, C/D box analysis revealed marked elevation in the number of UG dinucleotides and D-box sequences in NaF exposed HOS cells. Herein, we report miR-124 and miR-155 as the new possible players involved in the development of fluorosis. We show that the alterations in UG dinucleotides and D-box sequences of snoRNAs could be due to NaF exposure.

Here we present the results of a large-scale bioinformatics annotation of non-coding RNA loci in 48 avian genomes. Our approach uses probabilistic models of hand-curated families from the Rfam database to infer conserved RNA families within each avian genome. We supplement these annotations with predictions from the tRNA annotation tool, tRNAscan-SE and microRNAs from miRBase. We identify 34 lncRNA-associated loci that are conserved between birds and mammals and validate 12 of these in chicken. We report several intriguing cases where a reported mammalian lncRNA, but not its function, is conserved. We also demonstrate extensive conservation of classical ncRNAs (e.g., tRNAs) and more recently discovered ncRNAs (e.g., snoRNAs and miRNAs) in birds. Furthermore, we describe numerous "losses" of several RNA families, and attribute these to either genuine loss, divergence or missing data. In particular, we show that many of these losses are due to the challenges associated with assembling avian microchromosomes. These combined results illustrate the utility of applying homology-based methods for annotating novel vertebrate genomes.

Mammalian H/ACA RNPs are essential for ribosome biogenesis, premessenger RNA splicing, and telomere maintenance. These RNPs consist of four core proteins and one RNA, but it is not known how they assemble. By interrogating the site of H/ACA RNA transcription, we dissected their biogenesis in single cells and delineated the role of the non-core protein NAF1 in the process. NAF1 and all of the core proteins except GAR1 are recruited to the site of transcription. NAF1 binds one of the core proteins, NAP57, and shuttles between nucleus and cytoplasm. Both proteins are essential for stable H/ACA RNA accumulation. NAF1 and GAR1 bind NAP57 competitively, suggesting a sequential interaction. Our analyses indicate that NAF1 binds NAP57 and escorts it to the nascent H/ACA RNA and that GAR1 then replaces NAF1 to yield mature H/ACA RNPs in Cajal bodies and nucleoli.

RNA polymerase II transcribes genes encoding proteins and a large number of small stable RNAs. While pre-mRNA 3′-end formation requires a machinery ensuring tight coupling between cleavage and polyadenylation, small RNAs utilize polyadenylation-independent pathways. In yeast, specific factors required for snRNA and snoRNA 3′-end formation were characterized as components of the APT complex that is associated with the core complex of the cleavage/polyadenylation machinery (core-CPF). Other essential factors were identified as independent components: Nrd1p, Nab3p and Sen1p. Here we report that mutations in the conserved box D of snoRNAs and in the snoRNP-specific factor Nop1p interfere with transcription and 3′-end formation of box C/D snoRNAs. We demonstrate that Nop1p is associated with box C/D snoRNA genes and that it interacts with APT components. These data suggest a mechanism of quality control in which efficient transcription and 3′-end formation occur only when nascent snoRNAs are successfully assembled into functional particles.

In Saccharomyces cerevisiae, snoRNAs are encoded by independent genes and within introns. Despite this heterogenous organization, snoRNA biosynthesis relies on a common theme: entry sites for 5′–3′ and 3′–5′ exonucleases are created on precursor molecules allowing the release of mature snoRNAs. In independently transcribed snoRNAs, such entry sites are often produced by the Rnt1p endonuclease. In many cases, cleavage sites are absent in the 3′ portion of the pre-snoRNAs, suggesting that processing starts from the 3′ end of the primary transcript. Here we show that cleavage/polyadenylation sites driving efficient polyadenylation, such as CYC1, prevent production of mature and functional snoRNPs. With these sites, snoRNA accumulation is restored only if polyadenylation activity is inhibited. Analysis of sequences downstream of snoRNA-coding units and the use of strains carrying mutations in RNA polymerase II (polII) cleavage/polyadenylation activities allowed us to establish that formation of snoRNA mature 3′ ends requires only the cleavage activity of the polII 3′-processing machinery. These data indicate that, in vivo, uncoupling of cleavage and polyadenylation is necessary for an essential cellular biosynthesis.

The nucleolar localization elements (NoLEs) of U17 small nucleolar RNA (snoRNA), which is essential for rRNA processing and belongs to the box H/ACA snoRNA family, were analyzed by fluorescence microscopy. Injection of mutant U17 transcripts into Xenopus laevisoocyte nuclei revealed that deletion of stems 1, 2, and 4 of U17 snoRNA reduced but did not prevent nucleolar localization. The deletion of stem 3 had no adverse effect. Therefore, the hairpins of the hairpin–hinge–hairpin–tail structure formed by these stems are not absolutely critical for nucleolar localization of U17, nor are sequences within stems 1, 3, and 4, which may tether U17 to the rRNA precursor by base pairing. In contrast, box H and box ACA are major NoLEs; their combined substitution or deletion abolished nucleolar localization of U17 snoRNA. Mutation of just box H or just the box ACA region alone did not fully abolish the nucleolar localization of U17. This indicates that the NoLEs of the box H/ACA snoRNA family function differently from the bipartite NoLEs (conserved boxes C and D) of box C/D snoRNAs, where mutation of either box alone prevents nucleolar localization.